This invention relates to the specific inhibition of RNA by interaction with RNA.
The specific inhibition of genes by oligonucleotides, e.g. in order to achieve a therapeutic blocking of deregulated oncogenes or viral genes, is based on the ability of such complementary RNA or DNA so-called antisense-oligonucleotides to hybridise with mRNAs, processing signals or pre-mRNAs and in this way interrupt the transfer of information from genes to proteins.
The use of antisense DNA results in the breakdown of complementary target RNA by RNAse H-cleaving of the hybrid formed, resulting in the irreversible destruction of the complementary RNA.
When antisense RNA is used, a so-called hybrid arrest of translation or processing occurs, the RNA/RNA hybrids constituting the structural obstacle. It is assumed that such hybrids accumulate in the cells; their subsequent fate has not hitherto been investigated. As far as is known at present, this mechanism is largely thought to be a reversible event. The use of antisense-RNA molecules has the advantage that these molecules may either be synthesized in vitro and introduced into the cells or that the genes coding for them can be introduced into the cells so that the inhibiting RNA can be produced within the cell. However, nobody has hitherto succeeded in bringing such genes into a form which makes it possible to produce an effective quantity of antisense RNA in the cell.
Very recently, a third principle of RNA inhibition has been discovered and made available for use in vitro.
This principle is based on the ability of RNA molecules the so-called ribozymes, to recognise certain RNA sequences, bind to them and cleave them. It was derived from the autocatalytic cleavage reactions of RNA molecules in plant viroids and satellite RNA observed in vivo.
On the basis of certain structural requirements for the ribozyme catalysed RNA cleavage, it is now possible to construct de novo ribozymes which have an endonuclease activity directed in trans to a certain target sequence. Since these ribozymes, of which the ones which have been most carefully researched are known as hammer-head ribozymes on account of their structure, can act on numerous different sequences, the corresponding ribozyme can be xe2x80x9cmade to measurexe2x80x9d for virtually any RNA substrate. This makes ribozymes interesting and extremely flexible tools for inhibiting specific genes, with the result that they are a promising alternative to antisense constructs, which have already demonstrated potential therapeutic use.
One ribozyme model currently known which has so far been researched most thoroughly has three structural domains; on the basis of this model, ribozymes against CAT-mRNA have already been successfully constructed (Haseloff et al., 1988; Uhlenbeck et al., 1987):
The three domains comprise:
a) a highly conserved region of nucleotides flanking the cleavage site in the 5xe2x80x2 direction. This usually means the sequence GUC, although modification in the GUA or GUU also showed a substantially undiminished cleaving activity. Cleaving was also found after the sequences CUC, and to a lesser extent for AUC and UUC as well (the requirements for efficient cleaving have not yet been fully explained).
b) the highly conserved sequences contained in naturally occurring cleavage domains of ribozymes, forming a sort of base-paired stem;
c) the regions which flank the cleavage site on both sides and ensure the exact arrangement of the ribozyme in relation to the cleavage site and the cohesion of the substrate and enzyme (in the experiments carried out hitherto, 8 bases were selected on each side).
RNA enzymes can be constructed according to this model and have already proved suitable in vitro for the efficient and specific cleaving of RNA sequences (Haseloff et al., 1988).
Very recently, further types of autocatalytic RNA cleavage activity were discovered which may be used for the targeted RNA inhibition. One of these models is the so-called hairpin ribozyme, the active site of which is derived from the minus strand of the satellite RNA of tobacco ring spot virus (Hampel and Tritz, 1989). Other self-cleaving RNA activities are associated with hepatitis delta virus (Kuo et al., 1988; Sharmeen et al., 1988; Wu et al., 1989) and with RNAseP (Altman et al., 1988).
The experiments which preceded the studies for the present invention served to compare the activities of antisense RNA, antisense DNA and ribozymes. These experiments were carried out using the snRNP U7-dependant histone-preRNA-processing reaction, in that an in vitro system which processes U7-dependent histone-preRNA (Mowry et al., 1987, Soldati et al., 1988). It was found that antisense RNA is the most potent inhibitor, and the inhibition is reversible. The inhibitory effects of antisense DNA and of ribozymes of the hammer-head type, both of which are irreversible, were within the same order of magnitude, each requiring an approximately one thousand-fold excess over the substrate RNA to achieve total inhibition.
Whereas the previous tests had been carried out with ribozymes with naked RNA in protein-free systems, the preliminary tests for the present invention were the first experiments to demonstrate that synthetically produced ribozymes directed to a specific sequence show a cleaving activity even in a medium which contains protein. This fact provided a first indication of a potential use in vivo.
One of the limiting factors in the use of ribozymes to inhibit the expression of specific genes would be in the build-up of a ribozyme concentration which is sufficient to efficiently rule out a specific biological reaction; the reasons for this would be, as in the use of antisense RNA, the inadequate stability of the RNA, among other things.
The aim of the present invention was to provide a system for using mRNA as an in vivo inhibitor of mRNA, which overcomes the previous restrictions in the use of RNA, by making an effective concentration of inhibition RNA available in the cell.
This aim is achieved according to the invention by means of a genetic unit which contains the transcription unit required for transcription by polymerase III and a DNA coding for the RNA which inhibits RNA function, this DNA being arranged within the genetic unit in such a way that the inhibiting RNA is part of the polymerase III transcript.